Discussion

The objective of
the present study was to evaluate the importance of morphology and
morphometry for the characterization of Hepatozoon species that parasitize snakes. The dimensions and morphology of the five Hepatozoon
species analyzed are in agreement with the description reported in the
literature for those species. We evaluated linear and area data of the
gamonts and submitted them to multivariate analysis, which permitted us
to divide the species into three different populations (Fig. 2).

In most reports on Hepatozoon
parasitizing snakes, new species were described on the basis of the
length and width of the gamonts and schizonts and also of oocysts,
sporocysts and sporozoites (Pessoa & Biasi 1973). Also, in a recent
review article it was reported that many Hepatozoon species were created by simply considering a new infected host (Smith 1996).

The
literature reports that exist immature and mature gamonts and that
these developmental stages could be interpreted as different species
(Smith 1996). Snakes with actively merogony may present gamonts of
different sizes, with more slender forms representing an immature
precursor to the larger gamonts (Smith et al. 1994). In the present
paper, the specimens of the five studied species were homogeneous and
the differentiation between mature and immature forms was not observed.

Some reports have demonstrated that the same Hepatozoon species can infect different hosts (Hull & Camin 1960, Pessoa et al. 1971). Besides, the gamonts of a same species of Hepatozoon
can present slight morphologic changes depending on the host. Pessoa et
al. (1974) demonstrated that it was possible to transfer H. tupinambis of the lizard Tupinambis teguixin to the snake C. durissus terrificus. The transfer induced changes in the parasite so that in the lizard, Hepatozoon species produced erythrocyte deformations but this characteristic was gradually lost in the experimental infections.

The possibility that all of the Hepatozoon
species described as parasites of terrestrial snakes, which are
transmitted by mosquitoes, constitute a single species whose blood
forms vary with the vertebrate host has also been discussed in the
literature (Ball 1970, Pessoa et al. 1971).

The above considerations show that a controversy exists on this subject. Some investigators believe that several Hepatozoon
species exist while others discuss the possibility that it is a single
species presenting different morphologic patterns depending on the
infected host (Smith 1996).

Our results show three different populations, supporting the hypothesis of the existence of several species of Hepatozoon that can infect different species of snakes. The parasites found in H. gigas are significantly different among themselves and the two are different from the other studied parasites.

By comparing the present results with those reported in the literature about the Hepatozoon
parasites of the same snakes studied by us, we observe that
disagreement exists about the determination of the species. Our results
show that the gamonts of the species H. terzii, H. philodryasi and the Hepatozoon sp. of C. durissus terrificus
do not differ morphologically, as shown in other reports describing
these species (Sambon 1909, Carini 1910, Phisalix 1931).

We
also observed different patterns when we analyzed the changes induced
in the erythrocytes by the parasites. We observed that all parasites
induced changes, mainly in erythrocyte length, as well as nuclear
flattening (Table II) and the changes induced by H. cyclagrasi were more pronounced, with alterations of the erythrocyte and its nucleus in terms of all of the parameters studied. H. migonei induced
deformity of the length and width of the erythrocyte but not in its
area. This parasite also caused a complete distortion of the nucleus,
changing its length, width, area, and position. H. terzii did
not modify the area of the erythrocyte or of its nucleus, but induced
significant modifications in their lengths and widths. Hepatozoon sp. of C. durissus terrificus induced changes involving the area and length of the erythrocyte, and also flattening of the nucleus. H. philodryasi was the species that least modified the erythrocyte. Even so, it altered its area and the width of the nucleus.

The
results obtained by morphologic evaluation showed that, depending on
the considered variable, such as, for instance, area and width of the
parasite, we can separate five different species, whereas for another
variable, such as length of the nucleus, we can differentiate only two
populations (Table I).
Since we believe that the individual analysis of a variable is not
sufficient to characterize a species, we submitted all the data
obtained for each parasite to multivariate analysis, a procedure that
permitted us to characterize only three different populations (Fig. 2).

Current knowledge does not allow us to state whether H. terzii, H. philodryasi and Hepatozoon sp. of C. durissus terrificus are
a single species or whether they are closely related different species.
This characterization may be obtained after analysis of sporogonic and
esquizogonic stages also with the use of molecular techniques for
comparison of the respective DNAs. If it is proven that these are only
one species, the use of morphometry, a simple and inexpensive
technique, will be of fundamental importance for the characterization
of Hepatozoon species. However, if subsequent studies
demonstrate that the species are different, the technique used by us
cannot be used for species characterization, but can only be considered
as an additional methodology for the study of this group, associated
with morphologic studies of other evolutionary forms of Hepatozoon spp.

In
the present study we did not evaluate other evolutionary forms of the
parasites, which could contribute to a differentiation of populations.
However, Smith (1996) reported that Hepatozoon sp. exhibit a
high degree of plasticity for many features and therefore,
morphological and morphometric features of the oocysts stage must be
statistically significant in order for species to be described. Recent
studies have demonstrated that identification using molecular
techniques may contribute to solve this problem (Wozniak et al. 1994,
Smith et al. 1999).